Construction And Application Of RNAi Vectors Of Low Gossypol Content Seed Of Upland Cotton

Cotton is not only an important fiber crop,, but also a significant source of oil and protein. Protein ofcotton seed could be used as food resource of human and animals. However, gossypol in cotton plants,, apolyphenolic compound, is a major factor of the cotton plant’s self-defense system against insect pests anddiseases, but gossypol is toxic for human and animals. and it limited the utilization of cotton seed asprotein source of human and animals. Therefore, many attempts have been made to remove gossypol incotton seeds and keep gossypol in plants, which had became the research hot spot through traditionalbreeding and genetic engineering methods. Traditional breeding usually took too exhausting work and longperiod to achieve new variety. Alternatively, the plant genetic engineering technology is fast and efficientwhich have attracted more and more attention. RNA interference (RNAi) called post transcriptional genesilencing, is a biological process in which RNA molecules inhibit gene expression, typically by causing thedestruction of specific mRNAmolecules. As a highly efficient technology of gene silence, RNAi technologyhas been widely used in crop genetic improvement.,If we use this technique,it needs to construct RNAivector which can express double strained RNA, and then the vector is transformed into plants.In this research, A novel RNA interference vector was firstly constructed based on thepCIMBLA2301vector with a seed-specific promoter, cadinene synthase (CAD) and (+)-delta-cadinenehydroxylase (CYP706B1), then the vector is transformed into cotton through Agrobacterium-mediated.Method. Main results were as follows:An Arabidopsis embryo development post-specific gene AT3G27660was found by expressionanalysis. In this study, the2000bp5＇flanking sequence of AT3G27660gene was cloned by PCRfrom Arabidopsis. A new plant expression vector named pCOASP3was successfully constructed, in whichthe reporter gene GUS was drived by ASP3promoter. The vector was transformed into Arabidopsisthaliana by mediation of Agrobacterium, and transgenic plants were obtained from T0. Histochemicalanalysis of the transgenic plants showed that seeds, embryo were dyed,but the leaves, roots, stems werenot dyed, which indicated that the promoter was seed-specific and normally cannot promote geneexpression in other plant organization A cotton seed specific promoter α-globulin B gene promoter was Cloned by PCR amplification fromGossypium hirsutum and TEGP vector was constructed.The interference fragment sequences of CAD and CYP genes were obtained through PCRamplification by using specific primers designed based on the sequence information of NCBI. The RNAivectors driven by seed specific promoter GP and ASP3targeted CAD and CYP were constructed by usingthe intron of pHANNIBAL. In this study, we totally built the three RNAi vectors: pCGSIAY, pCA3SIAY,pCGSIACD, these RNAi vectors were transformed into cotton by Agrobacterium-mediated method andembryonic callus were induced,...